Three-dimensional choroidal contour mapping in healthy population

Purpose was to study 3-dimensional choroidal contour at choroidal inner boundary (CIB) and choroidal outer boundary (COB) in healthy eyes. Healthy eyes imaged on wide field swept-source optical coherence tomography were included. Delineation of CIB and COB was done based on our previously reported methods. Quantitative analysis of the surfaces of CIB and COB was based on analyzing best fit spherical radius (R) (overall and sectoral). One hundred and seven eyes of 74 subjects with a mean age of 46.4 ± 19.3 years were evaluated. Overall, R COB (mean ± SD: 22.5 ± 4.8 mm) < R CIB (32.4 ± 9.4 mm). Central sector had the least R at COB (7.2 ± 5.9 mm) as well as CIB (25.1 ± 14.3 mm) across all age groups. Regression analysis between R (CIB) and age (r = −0.31, r2 = 0.09) showed negative correlation (P < 0.001) and that between R (COB) and age was positive (r = 0.26, r2 = 0.07) (P = 0.01). To conclude, central sector is the steepest sector in comparison to all the other sectors. This is indicative of a prolate shape of choroidal contour at CIB and COB. Outer boundary of choroid is steeper than inner boundary across all age groups. However, with ageing, outer boundary becomes flatter and inner boundary becomes steeper.

Detailed history, vision assessment, external eye examination, slit lamp examination, intraocular pressure measurement and fundus examination was done to rule out any corneal/lenticular/anterior chamber/vitreal/retinal/ optic nerve/choroidal/scleral abnormality.The refractive error of the subjects and best-corrected visual acuity (BCVA) was checked.Eyes with refractive error > ± 2 D were excluded and only subjects with BCVA better than 20/25 were included in the study.Axial length was measured using an optical biometer (IOLMaster 700, Carl Zeiss Meditec, Jena, Germany) and eyes with axial length < 21 mm or > 26 mm were excluded.Dilated imaging on wide-field swept-source optical coherence tomography (SS-OCT) 12X12 mm on the Plex Elite 9000 device (Carl Zeiss Meditec, Dublin, CA) centred on the fovea was obtained.The quality of the scan was ensured by the in-built scoring system in the swept-source optical coherence tomography (SS-OCT) machine.A score out of 10 is rewarded by the machine for every scan.Scans with scores ≥ 6 (highlighted as green) were accepted for the analysis.Only eyes with a good quality, normal scan were accepted for this study.SS-OCT scans were exported as complete 8-bit volumes.Each OCT volume comprised 1024 B-scans and the resolution of each scan was 1024 X 1536.Patients with systemic diseases such as diabetes, hypertension, impaired renal function, thyroid disorders, vascular disorders were excluded.

Delineation of choroidal inner boundary (CIB) and choroidal outer boundary (COB)
Choroidal boundaries COB and CIB were obtained based on our previously reported methods 5 .In particular, initial CIB and COB estimate in each B scan were obtained based on residual network-based encoder-decoder deep learning architecture (ResUnet) which were subsequently stacked in 3D to perform volumetric smoothening to get the final boundary estimates.Volumetric smoothing was done to correct the abrupt deviations in boundary estimates within each B-scan and across consecutives B-scans.To smooth abrupt boundary changes across B-scans, robust locally estimated scatterplot smoothening (RLOESS) 6 was employed and to smooth minor deformations with and across B-scans tensor voting 7,8 was applied.This approach achieved a Dice coefficient of 97%, against manual segmentation, for both CIB and COB.

Methods for quantitative analysis of choroidal inner and outer boundaries (CIB and COB)
To evaluate the CIB and COB objectively, we performed quantitative analysis of the surfaces based on analyzing best-fit spherical radius (R) 9,10 .The first best-fit spherical radius is estimated for the overall surface to understand the overall curvature of the surface.The best-fit sphere radius (R) for the point is obtained using sphereFit MATLAB toolbox developed based on the least-squares regression.
where (a, b, c) indicates the center and R indicate the radius of the sphere 9 .
Subsequently, we estimated best-fit spherical radius for each sector i.e., for central, nasal, temporal, superior and inferior sector.Intuitively, flatter surface will have larger radius and vice versa.
We obtained sector-wise mean and standard deviation of R to understand the curvature changes in each sector/quadrant.In particular, five quadrants-central, nasal, temporal, superior and inferior are considered centered over fovea.The center of the fovea was manually selected by the grader looking at the en-face image obtained at the internal limiting membrane (ILM) of the retina.The central quadrant is circular centered around fovea with a radius of 1 mm and the rest of the quadrants are outside the central quadrant with a 90-degree separation.We generated the binary masks of these quadrants and superimposed on the radius, thickness and curvature maps to get the quadrant wise statistics.To facilitate the expert grading and analysis, we developed an inhouse MATLAB based graphical user interface (GUI) to view the choroidal surfaces, thickness map, curvatures maps and to generate respective spread sheets consisting of sector-wise statistics.Mean values of central sector choroidal thickness were used as subfoveal choroidal thickness (SFCT) (Fig. 1).

Statistical analysis
Data was represented as mean ± standard deviation (SD).Analysis of variance was used to compare the inner and outer choroidal spherical radius among different sectors (nasal, inferior, temporal, superior and central).Generalized estimating equation was employed to compare the choroidal parameters in different age groups (< 30, 30-44, 45-59, and ≥ 60 years).This was done as both eyes of a subset of the study subjects were included in the study.The choroidal parameters between right and left eye were compared using paired t-test.Regression analysis was used to assess the correlation between age, axial length, SFCT and radius of curvature (CIB and COB).P value ≤ 0.05 was considered statistically significant.

Results
A total of 107 eyes of 74 subjects (including 33 subjects with bilateral eyes) were analyzed.The study cohort had a preponderance of females (42 subjects) with remaining males (32 subjects).The mean age of the study cohort was 46.4 ± 19.3 years (range, 17-89 years).The mean (± SD) BCVA (logMAR) was 0.02 ± 0.06.Axial length ranged from 22.29 to 25.94 mm (mean ± SD: 24.1 ± 1.1 mm) whereas mean SFCT was 286.0 ± 48.2 µm.One hundred and seven eyes were categorized in four categories based on age.There were 25 eyes < 30 years of age, 32 eyes in the 30-44 years age category, 23 eyes in 45-59 years of age and 27 eyes ≥ 60 years.

Choroidal parameters
Spherical radius for CIB and COB were measured in all 5 sectors (nasal, inferior, temporal, superior and central) for the entire cohort.Moreover, spherical radius for both CIB and COB were also compared across different age groups in all sectors.
Comparison of sectoral CIB and COB parameters between right and left eye (studied on 33 subjects or 66 eyes) failed to show statistically significant difference between all sectors (all P values ≥ 0.05) except CIB superior sector (P = 0.04) as shown in Table 2. Marginal model using generalized estimating equations (GEE) approach was used to compare the CIB and COB parameters in view of the repeated measurements within the same eye and to account for inter-eye correlation for both eyes.There was a statistically significant difference while comparing spherical radius of CIB and COB in different sectors (all P values < 0.001).This difference was also evident across different age groups (< 30, 30-44, 45-59, and ≥ 60 years) as shown in Table 3.

Discussion
Using novel algorithm for choroidal contour mapping on healthy subjects, we noted a significant difference of radius of curvature between CIB and COB with R CIB > R COB.We report gradual decrease in radius of curvature in CIB and in contrary, an increase in COB with age.Our normative database show that the central sector was the steepest amongst all the sectors for both, CIB as well as COB.Assessment of correlation of SFCT with radius of curvature at COB and CIB revealed a positive correlation with R CIB and a negative correlation with R COB.We also noted an increase in axial length was correlated with a reduction in radius of curvature for both CIB and COB.
In healthy eyes, cornea has been shown to have a prolate shape i.e. central curvature is steeper than the periphery 11 .We noticed the same trend in choroidal curvature at choroidal inner boundary as well as choroidal outer boundary with center being the steepest sector.COB, i.e., choroidal curvature at the choroidoscleral www.nature.com/scientificreports/interface, has been shown to have a bowl shape contour at the posterior pole in healthy eyes in previous studies 12,13 .Some authors have called this same bowl shape as convex 12 and others have called it concave 13 when looking from inside out.Chen et al. have shown prolate retinal shape in emmetropic eyes 14 .However, these results were not reproducible, wherein Atchison et al. showed most emmetropic eyes possess oblate shape (steepening towards the periphery) 15 .Our quantitative sectoral analysis has a robust methodology which is superior to the    www.nature.com/scientificreports/qualitative assessments and showed a significantly lower radius of curvature in central sector (steep center/prolate shape) versus other sectors (nasal, temporal, superior, inferior) at CIB and COB.Myopic and hyperopic defocus is known to cause increase and decrease in choroidal thickness (CT) respectively which is both rapid and reversible upon removal of the inciting stimulus 16 .Animal models including chicks have demonstrated choroidal thinning with myopia progression which correlated well with the reduction in choroidal blood flow.The choroidal thinning however improved with the reversal of myopia 17 .Whether these changes in choroidal thickness translate into similar changes of choroidal contour is unclear at present.
On comparison of overall CIB versus COB, overall COB was steeper than CIB.On comparison of corresponding sectors, COB was still steeper than CIB in all sectors.On evaluation of relationship of age to the choroidal contour, it was found that with age choroidal contour becomes steeper at CIB and flatter at COB.This is interesting because, although overall CIB becomes steeper with age, the significant difference between COB and CIB (COB being steeper than CIB) is still maintained in all sectors and across all age groups.
On sectoral analysis of choroidal contour at CIB, the central CIB became significantly steeper with age, but CIB in temporal, nasal, superior and inferior quadrant did not change significantly with age.This shows that age related changes in choroidal contour at CIB were primarily in the central sector.Sectoral analysis of choroidal contour at COB demonstrated that flattening of contour with age was significant in inferior, temporal and central sectors.
With age, there is an increased biomechanical stiffness in the sclera.This stiffness also varies between regions with anterior sclera showing largest stiffness growth with advancing age and posterior sclera showing the least 18 .The gross shape of sclera also changes with ageing.In an OCT based study by Tun and associates, it was shown that the shape of peripapillary sclera changes as a function of age 19 .The anterior surface of sclera had a characteristic V-shape with the tip of the V pointing towards the orbit.This V shape was shown to become more prominent with age, worse vision, thinner cornea, greater axial length, lower CT in peripapillary area and deeper anterior lamina cribrosa.Changes in scleral structure as well as composition have been identified in human myopia and experimental animal myopia models 20 .With increase in axial length, scleral thickness decreases in the posterior globe segment 21 .Also, there is reduction in the collagen fibril diameter in human myopic eyes indicative of tissue remodeling with changes in axial length 22 .It is known that the mechanical stress due to distension of vitreous cavity in myopia leads to thinning and traction on the chorioretinal surface causing lacquer cracks, retinal tears, posterior staphyloma, choroidal neovascularization and other complications 23,24 .Similarly, shorter axial length in hypermetropia may cause complications such as angle closure glaucoma due to crowding of anterior segment structures 25 .Optical defocus in chick models has been shown to induce rapid changes in proteins in the retina or RPE that have previously been linked with inherited and age related ocular pathologies in humans 26 .It has been suggested that during development, choroidal shape and thickness influences the growth of sclera and length of the eye and thus play an important role in emmetropization of the eye 27 .It will be interesting to study the relationship of change in choroidal contour with various diseased states.
With increase in axial length, steepness was noted to increase at COB as well as CIB but it did not reach statistical significance.Interestingly, corneal radius of curvature increases as axial length increases i.e., cornea becomes flatter with increase in axial length 28 .
On evaluation of 33 subjects whose bilateral eyes were included in our dataset, it was shown that there was no significant difference between right eyes and left eyes in choroidal contour at CIB or COB.Previous studies on choroidal thickness have also demonstrated no significant interocular difference 29 .
The current study has certain limitations.Our sample size was small and a bigger sample size would have added more strength to the study.Only 12X12mm area of choroid was analyzed.Therefore, the peripheral choroid including the effect of vortex veins on choroidal contour was not studied.The choroidal contour especially the choroidoscleral interface does not always follow a smooth pattern and may have an inflection point or S-shaped or irregular contour 13 .Moreover, the localized effect of short posterior ciliary artery entry sites on COB was not studied.As this was a cross sectional study, we could not evaluate the long-term changes in healthy groups during follow up with age.
In conclusion, we report normative database for 3-dimensional choroidal contour mapping using novel algorithm and the changes in various age groups.In our future projects, we plan to study choroidal contour in different diseases such as high myopia, pachychoroid disease spectrum, AMD and compare with normative database.

Figure 1 .
Figure 1.Graphical user interface (GUI) demonstrating a 3-dimensional contour of choroidal inner boundary (CIB) and choroidal outer boundary (COB) which can be rotated with the cursor to understand the shape.In this case, the best fit spherical radius of curvature (R) at COB is 20.3 mm which is lesser than CIB of 31.1 mm implicating a steeper contour at COB.

Figure 2 .
Figure 2. Mean of overall best fit spherical radius of curvature (R) at choroidal inner boundary (CIB) and choroidal outer boundary (COB) in different age groups.The top of the diagram demonstrates that overall CIB is becoming steeper with age.R (CIB) is 35.4 mm, 33.6 mm, 32.2 mm and 28.3 mm in age groups < 30 years, 30-44 years, 45-59 years and ≥ 60 years respectively.The bottom of the diagram shows that overall COB is becoming flatter with age.R (COB) is 21.5 mm, 21.3 mm, 23.7 mm and 23.8 mm in age groups < 30 years, 20-45 years, 45-59 years and ≥ 60 years respectively.Only 2 groups (< 30 years and 45-59 years) are shown in this diagram as the values of < 30 years and 30-44 years were very close; and the values of 45-59 years and ≥ 60 years were very close to be appreciated on the small line diagram.

Table 1 .
Showing comparison of best fit spherical radius of curvature at choroid inner boundary (CIB, mm) and best fit spherical radius of curvature at choroid outer boundary (COB, mm) across different age groups.These values are mean radius of curvature in mm ± standard deviation (SD), (95% confidence interval (CI) is mentioned in the brackets; significant p values are highlighted in bold.

Table 2 .
Showing comparison of spherical radius of curvature at choroidal surfaces between right and left eye of 33 subjects (66 eyes).Significant p values are highlighted in bold.

Table 3 .
Shows the paired difference of spherical radius of curvature (in mm) in all the sectors (nasal, inferior, temporal, superior, central) at choroidal inner boundary (CIB) and choroidal outer boundary (COB) in different age categories.The paired difference of spherical radius of curvature (in mm) in all the sectors (nasal, inferior, temporal, superior, central) at choroidal inner boundary (CIB) and choroidal outer boundary (COB) in different age categories.It also shows overall (union of all five sectors) paired difference of spherical radius of curvature at CIB and COB.SD standard deviation, CI confidence interval; All measurements are in mm.Significant p values are highlighted in bold.

Table 4 .
Shows changes in spherical radius of choroidal inner boundary (CIB) and choroidal outer boundary (COB) with increase in age, axial length and subfoveal choroidal thickness (SFCT).